Phosphogypsum and poultry manure enhance diversity of soil fauna, soil fertility, and barley (Hordeum aestivum L.) grown in calcareous soils

Enrichment of calcareous soils with phosphogypsum and poultry manure amendments could increase nutrient availability, improve calcareous chemical characteristics, and enhance barley plant growth. In the current study, phosphogypsum (PG) and poultry manure (PM) were used to determine the effects of PG and PM application on soil fauna diversity, soil fertility, and barley yield. The pot experiment treatments were: C: control; PG1: 4.20 g kg−1 soil; PG2: 6.30 g kg−1 soil; PM1: 4.20 g kg−1 soil; PM: 6.30 g kg−1 soil, and their combinations. The results indicated that the application of PM alone or combined with PG had significant effects on the microbial biomass carbon (MBC), organic matter (OM), soil NPK availability, and yield of barley. Collembola and Prostigmata accounted for 50.0 and 43.3%, respectively, of the total number of soil fauna. Shannon and evenness indices increased significantly in the soil amended with PM alone or combined with PG. Amended soil with PG and/or PM significantly increased the yield and yield components of plants compared to the control. The PM1PG2 treatment increased the yield by 76.2% above the control.

In Egypt, the calcareous soils constitute about 27-36% of the total area. In calcareous soils where pH is high and CaCO 3 is dominant, plants suffer from low availability of P and K; therefore, increasing the availability of these nutrients is important. Cultivation of calcareous soils presents many challenges, such as low water holding capacity, high infiltration rate, poor structure, low organic matter (OM) and clay content, low cation exchange capacity (CEC), loss of nutrients via leaching or deep percolation, surface crusting, cracking, loss of nitrogen (N) fertilizers, low availability of phosphorous (P), and micronutrients [1][2][3][4] . At present, inorganic fertilizers are being intensively used to meet the increasing demand for agricultural products in these areas, thereby threatening to contaminate groundwater as a consequence of nitrate percolation. Thus, there is a need to find an appropriate soil management strategy that could reduce inorganic fertilization application and enhance the soil fertility in calcareous soils.
Nutrient-enriched poultry manure (PM) is commonly applied as a cost-effective alternative to inorganic fertilizers. The addition of PM to calcareous soils could be a promising strategy for maintaining OM and improving soil properties 5 . However, negative environmental impacts, including greenhouse gas emissions, are generally associated with PM applications 6 . It is therefore desirable to develop strategies to reduce greenhouse gas emissions from manure-applied soils. We hypothesized that the incorporation of PM into sandy calcareous soil may be another plausible alternative to inorganic fertilizers. Phosphogypsum (PG) is a by-product resulting from the production of phosphoric acid from rock phosphate. It is the major solid waste generated from the wet process, generating approximately 5 metric tons of waste per metric ton of phosphoric acid produced 7,8 . Recycling PG is very important for producers of phosphoric acid. Based on PG chemical and physical characteristics, the potential usage is soil conditioner or landfill cover 9 . As the hazardous effects of PG waste increase in the environment owing to continued industrialization, appropriate management is required to reduce the adverse impacts on the www.nature.com/scientificreports/ ecosystem 10 . The major challenge in Egypt is the accumulation of PM waste, which could cause pollution issues unless environmentally and financially sustainable management strategies are used. The addition of PG to the soil is one of the most important ways to get rid of it and benefit from improving the properties of different soils and increasing crop production. It is a source of many important nutrients for plants, such as Ca, P, S, and Fe; therefore, adding PG to the soil leads to supplying plants with nutrients during the different growth stages. Phosphogypsum is also used in the reclamation of sodic soil due to its rapid solubility, the liberation of Ca in the soil matrix, and the replacement of Na, which leads to increased water infiltration in the soil 11,12 . Microbial aggregates in the soil play a role in preserving organic matter and maintaining its fertility. Microbial biomass, and enzymatic activity are important indicators of the soil for agriculture. Soil fauna is an essential component of soil health because it performs vital ecosystem functions like the decomposition of organic matter and nutrient cycling 13,14 . Soil fauna depend on organic matter and soil nutrient availability 15,16 . Soil fauna activity improves soil structure, decomposes OM, and increases soil fertility [17][18][19] . Orbatid mite species are utilized as bioindicators of the soil in which they dwell because they are crucial to the biogenic stage of humification of plant wastes 14 .
Collembolans are regarded as environmentally friendly since they recycle and degrade agricultural waste in the soil, along with bacteria, nematodes, protozoa, and fungi 20 .
In light of these challenges and potential solutions, this study aims to evaluate the effect of PM and PG applications on some calcareous soil chemical properties and fertility. We hypothesize that adding PM and PG will improve the fertility of calcareous soils and enhance barely growth and productivity; thus, this research is significant in providing farmers with cost-effective and sustainable waste management.
The novelty of this study, PM amendment can effectively enhance soil fertility and crop production, but it cannot significantly improve the soil pH or increase soil fauna in calcareous soils. Therefore, the combination of PG and PM applications will not only lower soil pH in calcareous soils but also further improve soil fauna and increase its quality. To the best of our knowledge, there are few studies on the combination use of recycled PG with PM on improving soil quality and soil fauna in calcareous soils. Therefore, the aim of this study was to evaluate the effect of PG, PM, and their mixtures at different rates on improving soil fauna diversity, soil fertility, and the yield of barley grown in calcareous soil.

Materials and methods
Soil, Phosphogypsum, poultry manure characterization. The PG was obtained from a fertilizer industry factory in El-Sharkia Governorate, Egypt, while the PM was collected from a poultry farm using sawdust in the poultry litter for 40 days. A soil sample (0-20 cm) was collected from the Qetaa Maryout location (latitude 30.88°86°N, longitude 29.8°54°E), Amreya 1, Alexandria, Egypt. Organic matter (OM) in PG and PM was determined using the Walkley-Black method 21 . The pH of the soil and PG were measured in 1:2.5 (w/v ratio), and the electrical conductivity (EC) of the soil was measured in soil paste. Meanwhile, the pH and EC of PM were measured in a 1:10 (w/v) ratio using a pH meter and EC meter, respectively. Soluble calcium and magnesium in soil paste were determined using the versenate method, according to Page et al. 22 . The total N, P, and K were measured according to Sparks et al. 23 . Cation exchange capacity (CEC) was measured according to Page et al. 22 . Total calcium carbonate was determined by Collin's Calcimeter method, Nelson 24 . Available soil P was measured using a spectrophotometer and the ascorbic acid method after the soil had been extracted with a 0.50 M NaHCO 3 solution at a pH of 8.30 25 . Available soil N was measured using the Kjeldahel method after being extracted by a 2 M KCl solution 26 . Available soil K was determined using a flame photometer after extraction by 1.0 mol L −1 ammonium acetate at pH 7 27 . Microbial biomass carbon (MBC) was determined using 25 g of soil samples that were fumigated for 24 h at 250 °C with ethanol-free chloroform. After extracting the soil with K 2 SO 4 , the amount of extractable organic C was calculated using K 2 Cr 2 O 7 and H 2 SO 4 for 30 min at 170 °C and titrated against ferrous ammonium sulphate using ferroin as the indicator.
MBC was calculated as: where: EC = Extractable C; Kc = 0.38 (K c is the K 2 SO 4 extract efficiency factor 28 . Some chemical properties of the soil, PM and PG are shown in Table 1.

Soil fauna analysis.
At the end of the experiment, soil samples were taken from each pot, and the soils were air dried before being placed in a 4 °C refrigerator. Berlese's funnels 29 were used to extract soil fauna and microorganisms. Separated organisms were cleaned with lactic acid and kept in 70% ethanol before being identified 27,28 . PAST, a programme for analysing paleontological data, version 4.08, was used to estimate the Shannon diversity index (H′) and Pielou's evenness (J′). The Shannon index (H′) was derived as − Pi ln (Pi), where Pi is the ratio of the number of species i to the total numbers. Evenness (J′) index was calculated as: According to Abdellatif et al. 30 , the soil fertility index was calculated using the following equation: where FI is soil fertility index; FN, FP, and FK the available soil N, P, and K and FOM for the OM (%).

Statistical analysis.
A one-way ANOVA was used to identify the differences between various treatments.
Tukey post-hoc comparisons between the various groups were carried out if there were significant variations in the means. The obtained data were statistically analyzed using SAS 9.4 (SAS Institute Inc., Cary, NC).

Results
Barley yield and its components. The application of PG and PM significantly increased the spike weight, flag leaf area (FLA), 1000-grain weight, grain weight, and straw weight of the barely grown plants compared to the C treatment ( Table 2). The spike weight of barley plants ranged between 14.01 and 18.38 g per pot. The addition of PM, PM2, PM1PG1, PM1PG2, PM2PG1, and PM2PG2 to the soil increased the spike weight of the  −1 , and the highest grain weight was observed in PM1PG2 treatment, while the lowest value was in the C treatment. The grain weight of the barley plants increased by 22.9 and by 71.8% in PM1 and PM2, respectively, as compared to the C treatment (Table 2). However, the differences in grain weight between the PM2PG1, PM2PG2, and PM2 treatments were not statistically significant.
The 1000-grain weight of barley plants increased significantly with PM alone or with the PG addition ( Table 2). The 1000-grain weight of barley plants increased by 19.1 and 36.1% in PM1 and PM2 treatment, respectively, compared with the C treatment. The PM2PG1 treatment recorded the highest 1000-grain weight of barley compared to other treatments. The differences in 1000-grain weight among the PM2PG1, PM2PG2, and PM2 treatments were not statistically significant.
Soil chemical characteristics and fertility. The added PG and PM amendments to the soil had significant effects on the calcareous soil chemical properties, i.e. soil NPK availability, OM, and MBC contents. The application of PG and PM amendments increased the soil NPK availability, OM, and MBC values significantly as compared to the C treatment ( Table 3). The highest values of OM were observed in PM2PG2, while the lowest values were in the C treatment. The OM content of the soil increased by about 22.5 and 41.6% over the C in the PM1 and PM2 treatments, respectively. However, the differences in OM values between PM1PG2 and PM2PG1 were not statistically significant.
As shown in Table 3, the MBC values were significantly increased with the addition of the PM or PG amendments as compared to the C treatment. The MBC values increased from 2.16 mg kg −1 in the C treatment to 5.37 mg kg −1 in the PM1PG2. However, the differences in MBC among the PM1, PM2, PM1PG1, and PM2PG2 were not statistically significant. Compared to the C treatment, the MBC values increased with the addition of PG and PM to the soil.
In general, the available soil NPK was significantly increased by the addition of PM alone or combined with PG amendments ( Table 3). The concentrations of available soil NPK in the PM, PG, and their combinations were higher than the C treatment. Overall, the PM2PG2 treatment gave the highest soil available values of N and P, while the PM2PG1 treatment gave the highest soil available K values. Available soil N and P in PM1 and PM2 treatments were increased by 18.37%, 26.49%, 31.21%, and 45.71%, respectively, as compared to the C treatment. The addition of PM with PG increased soil available K from 248.19 to 266.92 mg kg −1 and to 257.51 mg kg −1 in PM1PG1 and PM1PG2 treatments. However, the difference in available soil P between PM2 and PM1PG1 treatment was not significant.

Soil fauna diversity.
In general, the amended soil with PG and PM had significant effects on the number of different soil microorganisms ( Table 4). The increase was more evident with the higher application rates for PG and PM. Overall, the PM1PG2 treatment gave the highest total numbers of soil fauna, followed by the PM2PG1 treatment, while the lowest values were found in the C treatment. The total number of soil microorganisms Table 3. Effect of PG and PM amendments on OM, MBC and soil availability of N, P and K. C Control, PM1 poultry manure at 4.2 g kg −1 soil, PM2 poultry manure at 6.30 g kg −1 soil, PM1PG1: poultry manure at 4.20 g kg −1 soil + phosphogypsum at 4.20 g kg −1 soil, PM1PG2: poultry manure at 4.20 g kg −1 soil + phosphogypsum at 6.30 g kg −1 soil, PM2PG1: poultry manure at 6.30 g kg −1 soil + phosphogypsum at 4.20 g kg −1 soil, PM2PG2: poultry manure at 6.30 g kg −1 soil + phosphogypsum at 6.30 g kg −1 soil. Each value represents a means ± standard error (SE) of five replicates. Biodiversity soil indices. The PM alone or in combination with the PG treatments considerably impacted the Evenness and Shannon's indices (Fig. 1). Overall, the PM1PG2 treatment gave the highest Evenness and Shannon's values, while the control treatment gave the lowest value. The evenness index ranged from 0.47 to 0.64 in the PM1 treatment and from 0.47 to 0.75 in the PM2 treatment. There were no statistically significant differences in the evenness index values between the PM1 and PM2PG2 or between the PM1PG2 and PM2PG2. Shannon's index values increased in the PM1 and PM2 treatments by 19.11 and 27.94%, respectively, compared to the control treatment.

Discussion
Soil fauna contribute significantly to soil quality by improving soil structure and decomposing organic matter 31 .
In this study, the total number of soil fauna, Shannon and evenness indices increased significantly when the soil was treated with PM alone or in combination with PG. Similarly, Seleem et al. 3 found that the soil fauna populations, Shannon, and evenness indices increased significantly in calcareous soil amended by PM alone or combined with vinasse. This result may be due to the high OM content of the PM. The increasing OM content of the soil increased the diversity of the total number of Collembola and Oribatid mites [32][33][34][35] . In PM addition alone or combined with PG, there was a positive correlation between the total number of soil fauna and OM content (R 2 = 0.82); soil fertility index (R 2 = 0.86); MBC (R 2 = 0.98). Islam et al. 31 found a significant positive correlation between the soil Collembola population and organic amendments. A positive correlation between soil C and the total number of soil fauna has also been reported [31][32][33] . This result coincides with that obtained by Rosildaet al. 34 Table 4. Effect of PG and PM amendments on number of different soil microorganisms. C Control, PM1 poultry manure at 4.2 g kg −1 soil, PM2 poultry manure at 6.30 g kg −1 soil; PM1PG1: poultry manure at 4.20 g kg −1 soil + phosphogypsum at 4.20 g kg −1 soil, PM1PG2: poultry manure at 4.20 g kg −1 soil + phosphogypsum at 6.30 g kg −1 soil, PM2PG1: poultry manure at 6.30 g kg −1 soil + phosphogypsum at 4.20 g kg −1 soil, PM2PiG2: poultry manure at 6.30 g kg −1 soil + phosphogypsum at 6.30 g kg −1 soil. Each value represents a means ± standard error (SE) of five replicates. www.nature.com/scientificreports/ who noticed that, the total numbers of Acari and Collembola species in soils increased with increasing soil OM content. Collembola represents one of the most abundant groups of soil-dwelling animals, playing a dominant role in the nutrient cycle, soil formation, and decomposition of OM 35,36 . Furthermore, the carbon content of the roots greatly affected the species formation of Oribatida mite, probably via rhizodeposition, as they feed on dead roots or root-associated fungi 37 . The increase in total number of soil fauna in soil amended by PM1PG2 treatment may be due to the higher OM and nutrient contents in the PM and PG (Table 1). Low total numbers of soil microorganisms in the C treatment may be due to the low C content 38 . The added PG and PM amendments to the soil had significant effects on the calcareous soil chemical properties, i.e. NPK availability, OM, and MBC. The results indicated that, the type and rates of the PG and PM amendments significantly affected the availability of NPK, OM, and MBC. The PM2PG2 treatment recorded the highest OM, available N, and available P compared with other treatments. The application of the PG and PM amendments increased nutrient availability, OM, and MBC significantly as compared to the untreated soil. The increase in OM due to the application of organic amendments was confirmed in several studies [39][40][41][42][43] . These results may be a direct effect of PG and PM additions on increasing the nutrient contents. In addition, there is an indirect effect via improvements in soil properties that induces optimal root and shoot growth. Organic matter plays a critical role in the soil ecosystem because it provides substrates for decomposing microbes, improves soil structure, and increases water holding capacity 39,43 . Soil OM is the principal indigenous source for soil available N; beside that, it contains about 65% of the total soil P and provides significant amounts of S and other nutrients essential for plant growth [44][45][46] . The interaction between PM and PG led to a highly significant increase in available soil P in the soil. These findings are expected because PG contains 0.70 to 1.0% P 2 O 5 . A similar trend was obtained with the available soil N in the interaction between PG and PM treatments, as PG can reduce the loss of N as NH 3 volatilization. Thus, more N derived from the PM was retained in the soil system, increasing the available soil N. These results may be a direct effect of PG and/or PM additions on increasing the nutrient contents. In addition, there is an indirect effect via improvements in soil properties that induces optimal root and shoot growth. The application of PM combined with PG reduces soil pH, enhances the availability of soil nutrients, and increases numbers of microorganisms. Similar results were reported by Gondek et al. 47 ; Chowdhury et al. 48 and Feng and Zhang 49 , who found that compost application increased soil OM and enhanced nutrient uptake through reduced nutrient leaching. The application of PG and PM gave the highest values of N, P, and K availability, confirming their ability to increase the efficient use of added fertilizers. However, this result may be attributed to the ability of added PG and/or PM to retain more nutrients, resulting in increased nutrient uptake. This result coincides with that obtained by Roudgarnejad et al. 50 , who postulate that the addition of different organic materials increases nutrient soil availability and uptake.
The addition of PG and/or PM significantly increased the barely yield and its components compared with the control treatment. A similar trend was reported by 51 , who reported that the application of organic amendments increased the plant height of barely planted significantly as compared to control. This may be due to the fact that the addition of PG and/or PM could increase nutrient release through direct application of amendments and/ or via increasing microbial activity that decomposes OM and thus increase soil fertility 51,52 . Amended soil with PG and/or PM greatly improves plant growth by increasing nutrient availability. Similar results were reported by 53,54 , who reported that organic amendments improved the soil quality.

Conclusions
Calcareous soils have low fertility and poor water holding capacity due to the lack of organic matter content. Phosphogypsum and poultry manure amendments significantly improved calcareous soil properties, increased the availability of NPK, microbial carbon content, and enhanced barely growth and yield. However, further field studies should be carried out to determine the proper poultry manure and phosphogypsum application rates to avoid a negative impact on the soil environment. In addition, studying the residual effect of PG and PM on yield, soil enzymes, and soil fauna diversity in long-term experiments to reduce the use of chemical fertilizers.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.